The present invention relates to a colorimetric assay method, apparatus and kit for the qualitative and quantitative determination of a bioactive polysaccharide in a product, more specifically, for the qualitative and quantitative determination of acemannan in an aloe-containing product.
Aloe is a tropical or subtropical plant characterized by lance-shaped leaves with jagged edges and sharp points. For centuries, this plant has been considered to have, and has been used for its, medicinal and therapeutic properties without any clear understanding or scientific analysis of the bases for such properties.
Because of the lack of knowledge about the aloe plant and its characteristics, most methods employed for the processing of the plant and its components result in end products which do not consistently contain the active ingredients. Traditional processes for the production of various aloe products typically involve crushing (pressure rollers, grinding e.g. use of a Thompson aloe leaf slitter), or pressing (TCS pressure extruder) of the entire leaf of the aloe plant to produce an aloe vera juice, followed by various steps of filtration and stabilization of the juice. The resulting solution is then incorporated in, or mixed with, other solutions or agents to produce the products which could be, for example, a cosmetic, a health food drink, or a topical ointment. Unfortunately, because of improper processing procedures, many of these so-called aloe products contain no active ingredients, namely, mucilaginous polysaccharides ("MP").
The principal disadvantage of such state of the art processes is the failure to recognize, and to take into account, that different components of the aloe leaf have characteristics that may not only be inconsistent with the intended use of the final product, but in many instances were deleterious to such use. Further, unless carefully controlled processes are used in processing the leaves of the aloe plant, the active ingredients, namely mucilaginous polysaccharides, of the leaves are destroyed during the process.
These active ingredients, the polysaccharides, have been identified, isolated and stabilized as described in U.S. Pat. Nos. 4,957,907 and 4,959,214, incorporated herein by reference. These active polysaccharides are hereinafter referred to as acemannan. Acemannan is an ordered linear polymer of substantially acetylated mannose monomers.
The fresh unpreserved gel is about 98.5-99.2% water. The total solid that remains after the water has been removed ranges from 0.8 to 1.5% of the total weight. The major constituents of that solid are mucilage, fiber, monosaccharides, polysaccharides, proteins, ash, fats, aloin and resin.
The physiological activity of acemannan and its pharmaceutical applications have been the object of numerous research studies at a number of laboratories, including Carrington Laboratories. These studies have primarily focused on the action of the activity of acemannan as an antiviral agent, an immunomodulator, a means of reducing opportunistic infections, and as a means of stimulating the healing processes.
Acemannan has been shown in laboratory studies to increase up to 300% in 48 hours the replication of fibroblasts in tissue culture which are known to be responsible for healing burns, ulcers and other wounds of the skin and of the gastrointestinal lining.
Acemannan has also been shown to increase DNA synthesis in the nucleus of fibroblasts. The increase in DNA synthesis in turn increases the rate of metabolic activity and cell replication which are fundamental steps in the healing process.
Acemannan has been shown in controlled studies to increase the rate of healing in animals as well, such as an effective treatment for gastric ulcers in animal studies. Over a three year period, laboratory rats, the stomachs of which react similarly to that of humans, were tested. Acemannan was found to be equivalent to or superior to current medications used for the treatment of gastric ulcers. Most such products act to inhibit hydrochloric acid in the stomach. Acemannan, however, works on a different principle and does not alter the natural flow of digestive acids. More recently, drinks containing extract of aloe vera plant have been placed on the market for consumers.
Several biochemical, inorganic and trace metal constituents have been associated with the plant extract. Prior to, and even after the discovery of active polysaccharides in aloe, various of these inorganic constituents have been claimed to be responsible for the "healing" power of the plant. As a result, manufacturers "process" the Aloe vera plant in different ways to suit their own beliefs and understanding. Thus, the scientific literature contains many contradictory opinions as to what components in the aloe vera plant are responsible for these "healing" effects. These contradictions have led to various methods of processing the aloe vera plant and to difficulty in establishing a reliable and easy to use composition standard for aloe vera plant and products. In some cases, the process used to process the aloe vera removes all of the polysaccharides and most of the other organic components of the plant, a process which causes the product to loose its biological activities, its characteristic aloe taste and its natural aloe texture. In such instances, the manufacturers try to reform the aloe texture by adding flavoring agents and non-aloe polysaccharides such as maltodextrin, dextran, plant gums, dextrose, etc. to the products. Unfortunately, however, these adulterated products can still be certified as "aloe vera products," whether or not they contain the acemannan.
Because of the large number in the market of these adulterated products with no acemannan, several attempts have been made over the years to develop a standard assay to measure the presence of active ingredients, such as acemannan, present in the aloe products. A comprehensive effort at developing a chemical component profile of aloe was made by the International Aloe Science Council (IASC) in 1982-1984. Some thirty parameters were considered relative to the verification of authentic aloe vera products. Out of these more than thirty parameters, four tests were commonly used. These four include percent of total solids, calcium, magnesium and high performance liquid chromatography (HPLC) peak ratios. The recommended standard, however, has inherent problems because three of the four tests, namely, total solid, calcium and magnesium can be found at varying levels in extracts of many other plants and fruits. The fourth test, "HPLC ratio" surprisingly is based on the presence of barbaloin, an anthraquinone derivative found in Aloe sap. Since aloe drink, which is meant for human consumption, is supposed to be substantially free of anthraquinones, the use of the HPLC ratio as a verification means of active ingredients of aloe in a drink containing aloe extract is inappropriate.
Another study to find an acceptable standard for the active ingredient of aloe vera has been conducted at Texas A&M University. Preliminary results of that study have been published in the SOFW--Journal, 119,255-268 (1993). Apart from the measurement of total solids, magnesium, calcium, potassium and sodium, an "HPLC profile" of aloe extract was obtained using an amino bonded column and a phosphate buffered acetonitrile as the eluant. The publication gives detailed information about an HPLC Profile peak known as "E-Peak." The "E-Peak" is not yet structurally characterized and has not been identified as possessing any bioactivity. Moreover, the HPLC retention time is highly variable and poses analytical problems with aloe drinks which may contain additives and preservatives.
Another assay method to measure polysaccharides of aloe vera involves the steps of initially precipitating the active polysaccharides with alcohol to get rid of alcohol soluble complexes of organic acids, inorganic salts and others. The precipitated polysaccharide is then measured by Dubois assay for total hexose. This assay is also problematic because most polysaccharides such as guar gum, dextran, dextrin, locust bean gum, and others would precipitate in alcohol as well as react positively with Dubois assay for hexose. Thus, products that have been adulterated with these non-aloe polysaccharides could still be certified as products containing the active ingredients of aloe vera.
Yet another assay method that is used is the HPLC Size Exclusion Chromatography (SEC). This is an "in-house" quality control procedure that is effective in determining the quality of the aloe vera product where no attempt to defraud has been made. However, it is difficult to use this procedure to regulate commercial products containing aloe vera.
Therefore, it is seen that there is a need for a reliable, sensitive, simple, specific and relatively foolproof validating assay methodology, apparatus and kit for determining the presence, and the amount, of naturally occurring bioactive polysaccharides found in aloe vera. The present invention is directed to such a methodology, apparatus, and kit.
Congo Red is an indicator primarily used for estimating free mineral acids and also for staining biological samples. Light absorption maximum of Congo Red in 1% w/v aqueous solution is approximately 488 nanometers wavelength. While studying the antitumor activity of Lentinan, a glucan from Lentinus Edodes, Sasaki et al., Gann, 67, 191-195 (1976), observed that the absorption maximum of Congo Red shifted toward longer wavelength in the presence of a large molecular weight lentinan polysaccharide. Small molecular weight fractions of the lentinan did not cause a shift in the absorption wavelength. Ogawa et al., Carbohydrate Research, 29, 397-403 (1973), reported the formation of a complex between gel-forming D-glucans and Congo Red in alkaline solutions. They postulated that complex formation of polysaccharide with Congo Red was due to orderly conformation of the molecule. However, there was no attempt to investigate whether complex formation was solely dependent on molecular size or whether other micromolecular characteristics of the polysaccharides were more important. Moreover, there was no report of non-glucan polysaccharides demonstrating shift in absorption with Congo Red. Most importantly, there was no attempt to use the Congo Red interaction with the polysaccharides to quantify these bioactive polysaccharides.